Structural requirements for the complement regulatory activities of C4BP

C4b-binding protein (C4BP) is a regulator of the classical complement pathway C3 convertase (C4bC2a complex). It is a disulfide-linked polymer of seven alpha-chains and a unique beta-chain; the alpha- and beta-chains are composed of eight and three complement control protein (CCP) domains, respectively. To elucidate the importance of the polymeric nature of C4BP and the structural requirements for the interaction between C4b and the alpha-chain, 19 recombinant C4BP variants were created. Six truncated monomeric variants, nine polymeric variants in which individual CCPs were deleted, and finally, four variants in which double alanine residues were introduced between CCPs were functionally characterized. The smallest truncated C4BP variant still active in regulating fluid phase C4b comprised CCP1-3. The monomeric variants were less efficient than polymeric C4BP in degrading C4b on cell surfaces. All three N-terminal CCP domains contributed to the binding of C4b and were important for full functional activity; CCP2 and CCP3 were the most important. The spatial arrangements of the first CCPs were found to be important, as introduction of alanine residues between CCPs 1 and 2, CCPs 2 and 3, and CCPs 3 and 4 resulted in functional impairment. The results presented here elucidate the structural requirements of individual CCPs of C4BP, as well as their spatial arrangements within and between subunits for expression of full functional activity.     

Structural requirements of anticoagulant protein S for its binding to the complement regulator C4b-binding protein

The vitamin K-dependent anticoagulant protein S binds with high affinity to C4b-binding protein (C4BP), a regulator of complement. Despite the physiological importance of the complex, we have only a patchy view of the C4BP-binding site in protein S. Based on phage display experiments, protein S residues 447-460 were suggested to form part of the binding site. Several experimental approaches were now used to further elucidate the structural requirements for protein S binding to C4BP. Peptides comprising residues 447-460, 451-460, or 453-460 of protein S were found to inhibit the protein S-C4BP interaction, whereas deletion of residues 459-460 from the peptide caused complete loss of inhibition. In recombinant protein S, each of residues 447-460 was mutated to Ala, and the protein S variants were tested for binding to C4BP. The Y456A mutation reduced binding to C4BP approximately 10-fold, and a peptide corresponding to residues 447-460 of this mutant was less inhibitory than the parent peptide. A further decrease in binding was observed using a recombinant variant in which a site for N-linked glycosylation was moved from position 458 to 456 (Y456N/N458T). A monoclonal antibody (HPSf) selective for free protein S reacted poorly with the Y456A variant but reacted efficiently with the other variants. A second antibody, HPS 34, which partially inhibited the protein S-C4BP interaction, reacted poorly with several of the Ala mutants, suggesting that its epitope was located in the 451-460 region. Phage display analysis of the HPS 34 antibody further identified this region as its epitope. Taken together, our results suggest that residues 453-460 of protein S form part of a more complex binding site for C4BP. A recently developed three-dimensional model of the sex hormone-binding globulin-like region of protein S was used to analyze available experimental data.     

Molecular characterization of the interaction between porins of Neisseria gonorrhoeae and C4b-binding protein

Neisseria gonorrhoeae, the causative agent of gonorrhea, is a natural infection only in humans. The resistance of N. gonorrhoeae to normal human serum killing correlates with porin (Por)-mediated binding to the complement inhibitor, C4b-binding protein (C4BP). The entire binding site for both porin molecules resides within complement control protein domain 1 (CCP1) of C4BP. Only human and chimpanzee C4BPs bind to Por1B-bearing gonococci, whereas only human C4BP binds to Por1A strains. We have now used these species-specific differences in C4BP binding to gonococci to map the porin binding sites on CCP1 of C4BP. A comparison between human and chimpanzee or rhesus C4BP CCP1 revealed differences at 4 and 12 amino acid positions, respectively. These amino acids were targeted in the construction of 13 recombinant human mutant C4BPs. Overall, amino acids T43, T45, and K24 individually and A12, M14, R22, and L34 together were important for binding to Por1A strains. Altering D15 (found in man) to N15 (found in rhesus) introduced a glycosylation site that blocked binding to Por1A gonococci. C4BP binding to Por1B strains required K24 and was partially shielded by additional glycosylation in the D15N mutant. Only those recombinant mutant C4BPs that bound to bacteria rescued them from 100% killing by rhesus serum, thereby providing a functional correlate for the binding studies and highlighting C4BP function in gonococcal serum resistance.     

Complement genetic markers in schizophrenia: C3, BF and C6 polymorphisms.

Polymorphic variants of C3, BF and C6 complement factors have been investigated in schizophrenic patients subdivided according to the existence or not of a family history of both schizophrenia and other psychiatric disorders. To analyze the contingency tables, besides the usual methods, log-linear models have been fitted. Significant associations have been found in the C3 system, with a decrease of C3*F in patients (contradicting previous findings), and in the BF system, with a decrease of FS phenotype among patients (confirming some previous results). No association has been found for the C6 polymorphism (in accordance to previous results). Therefore, the present findings only partially confirm previous results and do not clarify the relationship between complement genetic markers and schizophrenia, stressing some statistical difficulties.     

Bf and C3 complement types in rheumatoid arthritis

Bf and C3 complement types were studied in 100 male and 100 females patients from northern Sweden with erosive rheumatoid arthritis (RA) and compared with population controls. A significantly decreased frequency of the Bf FS phenotype was found particularly in males and in patients with a family history of polyarthritis. Significant Bf associations were also found with a more severe form of RA (functional classes III and IV) and with high titers of the rheumatoid factor. No significant difference with respect to C3 phenotype and gene frequencies was found between RA patients and controls. Thus, the association between RA and C3F found in some previous investigations was not confirmed.     

Ionic binding of C3 to the human pathogen Moraxella catarrhalis is a unique mechanism for combating innate immunity

Moraxella catarrhalis ubiquitous surface proteins A1 and A2 (UspA1/A2) interfere with the classical pathway of the complement system by binding C4b-binding protein. In this study we demonstrate that M. catarrhalis UspA1 and A2 noncovalently and in a dose-dependent manner bind both the third component of complement (C3) from EDTA-treated serum and methylamine-treated C3. In contrast, related Moraxella subspecies (n = 13) or other human pathogenic bacteria (n = 13) do not bind C3 or methylamine-treated C3. Experiments with recombinant proteins and M. catarrhalis mutants devoid of UspA1/A2 revealed that UspA1/A2 exert their actions by absorbing and neutralizing C3 from serum and restrain complement activation. UspA2 was responsible for most of the effect, and the Moraxella mutant lacking UspA2 was more sensitive to the lytic effect of human serum compared with the wild type. Interestingly, among the large number of bacteria analyzed, only M. catarrhalis has this unique ability to interfere with the innate immune system of complement by binding C3.     

APC mutation in the alternatively spliced region of exon 9 associated with late onset familial adenomatous polyposis

Germ-line mutations in the adenomatous polyposis coli (APC) gene are responsible for familial adenomatous polyposis (FAP). Genotype-phenotype correlation studies in patients with FAP have demonstrated associations of certain variants of the disease with mutations at specific sites within the APC gene. In a large FAP family, we identified a frameshift mutation located in the alternatively spliced region of exon 9. Phenotypic studies of affected family members showed that the clinical course of FAP was delayed, with gastrointestinal symptoms and death from colorectal carcinoma occurring on average 25 and 20 years later than usual, respectively. The numbers of colorectal adenomas differed markedly among affected individuals and the location of colorectal cancer lay frequently in the proximal colon. Our findings suggest that the exon 9 mutation identified in the pedigree is associated with late onset of FAP. The atypical phenotype may be explained by the site of the mutation in the APC gene. Analysis of the APC protein product indicated that the exon 9 mutation did not result in a detectable truncated APC protein. Given the location of the mutation within an alternatively spliced exon of APC, it is conceivable that normal APC proteins are produced from the mutant allele by alternative splicing.     

Concanavalin A variants of the fetoacinar pancreatic protein in the developing human pancreas and other biological sources

Two new sources for the fetoacinar pancreatic protein (FAP protein) are described in this study: amniotic fluids taken at 18 weeks gestation, and pancreatic juices from patients with pancreatic pathology. The FAP protein from different biological sources showed two kinds of molecular heterogeneity: (a) molecular weight, and (b) lectin-binding affinity. By Western blot the protein was shown to exist either as a doublet (the higher-Mr component at 110 kDa and the second in the range 80-100 kDa) or as a single band (110 kDa) depending on the source. By chromatography on Con A-Sepharose the protein could be separated into two variants, reactive and nonreactive. Most of the protein was present as the Con A-nonreactive variant. The Western-blot patterns of both variants in a given sample were identical. The FAP protein expression had an oncodevelopmental character; maximal concentration was seen in middle-gestation fetal pancreas extracts. Expression of the FAP protein Con A variants followed the same developmental pattern as that of total FAP protein, and their relative amounts remained almost constant during fetal growth. Evidence is given for the presence of lectin and molecular-weight heterogeneities of the protein as well as for the lack of a developmental pattern for the expression of these variants.     

Polymorphisms of serum proteins in Japanese patients with vascular diseases. I. Factor XIIIB, plasminogen and complement types in primary varicose veins.

The polymorphisms of the B subunit of coagulation factor XIII (F13B), plasminogen (PLG), complement C6, C7, factor B (BF) and factor I (IF) were studied among 21 unrelated Japanese patients with primary varicose veins (PVV) by isoelectric focusing followed by immunoblotting. The allele frequencies for F13B*2 and IF*A in PVV patients were significantly higher (F13B*2, p = 0.0047; IF*A, p = 0.0006) than those in healthy controls (n = 60). Significant associations of F13B 2 allotype [p = 0.0220, relative risk (RR) = 13.9] and IF A allotype (p = 0.0006, RR = 10.0) with PVV were observed; however, no significant association of PLG, C6, C7 or BF allotype with the disease was found.     

Association of genetic variants rs641153 (CFB), rs2230199 (C3), and rs1410996 (CFH) with age-related macular degeneration in a Brazilian population

This study aimed to investigate the association among genetic variants of the complement pathway CFB R32Q (rs641153), C3 R102G (rs2230199), and CFH (rs1410996) with age-related macular degeneration (AMD) in a sample of the Brazilian population. In a case-control study, 484 AMD patients were classified according to the clinical age-related maculopathy grading system (CARMS) and compared to 479 unrelated controls. The genetic variants rs1410996 of complement H (CFH), rs641153 of complement factor B (CFB), and rs2230199 of complement 3 (C3) were evaluated through polymerase chain reaction (PCR) and direct sequencing. The associations between single nucleotide polymorphisms (SNPs) and AMD, adjusted by age, were assessed by using logistic regression models. A statistically significant association was observed between AMD risk and rs2230199 variant with an OR of 2.01 (P  = 0.0002) for CG individuals compared to CC individuals. Regarding the comparison of advanced AMD versus the control group, the OR was 2.12 (P = 0.0036) for GG versus AA genotypes for rs1410996 variant. Similarly, the OR for rs2230199 polymorphism was 2.3034 (P  = 5.47^e-05) when comparing CG individuals to CC carriers. In contrast, the rs641153 variant showed a significant protective effect against advanced AMD for GA versus GG genotype (OR = 0.4406; P  = 0.0019). When comparing wet AMD versus controls, a significant association was detected for rs1410996 variant (OR = 2.16; P  = 0.0039) comparing carriers of the homozygous GG versus AA genotype, as well as in the comparisons of GG (OR = 3.0713; P  = 0.0046) and CG genotypes (OR = 2.2249; P  = 0.0002) versus CC genotype for rs2230199 variant, respectively. The rs641153 variant granted a significant protective effect against wet AMD for GA versus GG genotypes (OR = 0.4601; P  = 0.0044). Our study confirmed the risk association between rs2230199 and rs1410996 variants and AMD, and the protective role against AMD for rs641153 variant.     

Complement factor C4 in schizophrenia

The complement factor C4 was studied in 165 schizophrenic patients and in 330 controls. A highly significant increase in the frequency of C4B deficiency (BQO) was found among the schizophrenic patients compared with controls (p less than 0.0005).     

Native state hydrogen exchange study of suppressor and pathogenic variants of transthyretin

Transthyretin (TTR) is an amyloidogenic protein whose aggregation is responsible for numerous familial amyloid diseases, the exact phenotype being dependent on the sequence deposited. Many familial disease variants display decreased stability in vitro, and early onset pathology in vivo. Only subtle structural differences were observed upon crystallographic comparison of the disease-associated variants to the T119M interallelic trans-suppressor. Herein three human TTR single amino acid variant homotetramers including two familial amyloidotic polyneuropathy (FAP) causing variants (V30M and L55P), and a suppressor variant T119M (known to protect V30M carriers from disease by trans-suppression) were investigated in a residue-specific fashion by monitoring (2)H-(1)H exchange employing NMR spectroscopy. The measured protection factors for slowly exchanging amide hydrogen atoms reveal destabilization of the protein core in the FAP variants, the core consisting of strands A, B, E and G and the loop between strands A and B. The same core exhibits much slower exchange in the suppressor variant. Accelerated exchange rates were observed for residues at the subunit interfaces in L55P, but not in the T119M or V30M TTR. The correlation between destabilization of the TTR core strands and the tendency for amyloid formation supports the view that these strands are involved in amyloidogenicity, consistent with previous (2)H-(1)H exchange analysis of the WT-TTR amyloidogenic intermediate.     

Mannose Binding Lectin and Susceptibility to Rheumatoid Arthritis in Brazilian Patients and Their Relatives

Introduction

Rheumatoid arthritis (RA) is a commonly occurring systemic inflammatory auto immune disease and is believed to be associated with genetic factors. The innate immune complement protein Mannose binding lectin (MBL) and their MBL2 genetic variants are associated with different infectious and autoimmune diseases.

Methods

In a Brazilian cohort, we aim to associate the functional role of circulating MBL serum levels and MBL2 variants in clinically classified patients (n = 196) with rheumatoid arthritis including their relatives (n = 200) and ethnicity matched healthy controls (n = 200). MBL serum levels were measured by ELISA and functional MBL2 variants were genotyped by direct sequencing.

Results

The exon1+54 MBL2*B variant was significantly associated with an increased risk and the reconstructed haplotype MBL2*LYPB was associated with RA susceptibility. Circulating serum MBL levels were observed significantly lower in RA patients compared to their relatives and controls. No significant contribution of MBL levels were observed with respect to functional class, age at disease onset, disease duration and/or other clinical parameters such as nodules, secondary Sjögren syndrome, anti-CCP and rheumatoid factor. Differential distribution of serum MBL levels with functional MBL2 variants was observed in respective RA patients and their relatives.

Conclusions

Our results suggest MBL levels as a possible marker for RA susceptibility in a Brazilian population.     

Binding of complement inhibitor C4b-binding protein contributes to serum resistance of Porphyromonas gingivalis

The periodontal pathogen Porphyromonas gingivalis is highly resistant to the bactericidal activity of human complement, which is present in the gingival crevicular fluid at 70% of serum concentration. All thirteen clinical and laboratory P. gingivalis strains tested were able to capture the human complement inhibitor C4b-binding protein (C4BP), which may contribute to their serum resistance. Accordingly, in serum deficient of C4BP, it was found that significantly more terminal complement component C9 was deposited on P. gingivalis. Moreover, using purified proteins and various isogenic mutants, we found that the cysteine protease high molecular weight arginine-gingipain A (HRgpA) is a crucial C4BP ligand on the bacterial surface. Binding of C4BP to P. gingivalis appears to be localized to two binding sites: on the complement control protein 1 domain and complement control protein 6 and 7 domains of the alpha-chains. Furthermore, the bacterial binding of C4BP was found to increase with time of culture and a particularly strong binding was observed for large aggregates of bacteria that formed during culture on solid blood agar medium. Taken together, gingipains appear to be a very significant virulence factor not only destroying complement due to proteolytic degradation as we have shown previously, but was also inhibiting complement activation due to their ability to bind the complement inhibitor C4BP.     

Inherited deficiency of the sixth component of complement: a silent or null gene

Four families have been studied, some members of which have inherited deficiency of the sixth component of complement. The genetically determined electrophoretic variants of C6 were evaluated in all family members. Seven individuals were found who did not have the variant found in the serum of the parent from whom they inherited the deficiency. It is inferred that the isolated low levels of C6 in these individuals results from the heterozygous state of a normal C6 variant gene and a silent or null C6 gene; the genes determining electrophoretic variants and the low serum levels of C6 are allelic.     

Exploring the expression bar code of SAA variants for gastric cancer detection

We reported an integrated platform to explore serum protein variant pattern in cancer and its utility as a new class of biomarker panel for diagnosis. On the model study of serum amyloid A (SAA), we employed nanoprobe‐based affinity mass spectrometry for enrichment, identification and quantitation of SAA variants from serum of 105 gastric cancer patients in comparison with 54 gastritis patients, 54 controls, and 120 patients from other cancer. The result revealed surprisingly heterogeneous and most comprehensive SAA bar code to date, which comprises 24 SAA variants including SAA1‐ and SAA2‐encoded products, polymorphic isoforms, N‐terminal–truncated forms, and three novel SAA oxidized isotypes, in which the variant‐specific peptide sequence were also confirmed by LC‐MS/MS. A diagnostic model was developed for dimension reduction and computational classification of the 24 SAA‐variant bar code, providing good discrimination (AUC = 0.85 ± 3.2E?3) for differentiating gastric cancer group from gastritis and normal groups (sensitivity, 0.76; specificity, 0.81) and was validated with external validation cohort (sensitivity, 0.71; specificity, 0.74). Our platform not only shed light on the occurrence and modification extent of under‐represented serum protein variants in cancer, but also suggested a new concept of diagnostic platform by serum protein variant profile.     

A Trans-acting Factor May Modify Age at Onset in Familial Amyloid Polyneuropathy ATTRV30M in Portugal

Although all familial amyloid polyneuropathy (FAP) ATTRV30M patients carry the same causative mutation, early (<40) and late-onset forms (≥50 years) of FAP may coexist in the same family. However, this variability in age at onset is still unexplained. To identify modifiers closely linked to the TTR locus that may in part be associated with age at onset of FAP ATTRV30M, in particular in a group of very early-onset patients (≤30 years) when compared with late-onset individuals. A clinical genetic study at a referral center comprising a sample of 910 Portuguese individuals includes 589 Val30Met carriers, 102 spouses, and 189 controls from the general population. Haplotype analysis was performed, using eight intragenic single nucleotide polymorphisms (SNPs) at the TTR locus. We compared haplotypes frequency in FAP samples and controls and in parent-offspring pairs using appropriated statistical analysis. Haplotype A was the most common in the general population. Noteworthy, haplotype C was more frequent in early-onset (<40) than in late-onset patients (≥50 years) (p = 0.012). When comparing allelic frequencies of each SNP within haplotype C between “very early” (≤30 years) and late-onset (≥50 years) cases, the A allele of rs72922947 was associated with an earlier onset (p = 0.009); this remained significant after a permutation-based correction. Also, the heterozygous genotype (GA) for this SNP was associated with a decrease in mean age at onset of 8.6 years (p = 0.014). We found a more common haplotype (A) linked to the Val30Met variant and a possible modulatory trans effect on age at onset. These findings may lead to potential therapeutical targets.     

C4 urernic variant: An acquired C4 allotype

The major histocompatibility complex (MHC)-linked complotype region includes alleles for B, C2, and C4 loci. These loci are closely linked to each other and to HLA-DR on chromosome 6. The duplicated C4 loci,, C4A and B, are especially polymorphic. In seven patients with renal insufficiency, we observed a C4 variant with electrophoretic mobility between C4B2 and C4B3. Four of these patients were detected during a study of MHC markers in mernbranoproliferative glomerulonephritis. Complete complotype and HLA data from families of five of the seven patients demonstrated that the variant was not inherited. The pattern was revealed by immunofixation electrophoresis and also by C4-specific hemolytic overlay. In serial plasma specimens taken from one patient, the C4 variant appeared only after the patient became uremic. However, the variant could not be produced in normal plasma after incubation with C4-depleted uremic plasma. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions of immunoprecipitated C4 from these patients showed C4A and C4B chains of normal molecular mass; incompletely processed forms of C4 were not observed. We believe that this variant is probably acquired in the presence of uremia and may represent the C4B2.9 allele found by Wank and co-workers in many patients with glomerulonephritis. Family studies are mandatory to distinguish genetic variants from acquired alterations in the C4 phenotype.     

Identification of carriers of mutant prealbumin gene associated with familial amyloidotic polyneuropathy type I by Southern blot procedures: study of six pedigrees in the Arao district of Japan

Summary Fifty-six Japanese individuals from six pedigrees with familial amyloidotic polyneuropathy (FAP), together with 2 individuals with symptomatic FAP from an unknown pedigree were analyzed, using the Southern blot procedures for the prealbumin gene structure. A human prealbumin cDNA was used as the probe. Altogether, these individuals included 20 with symptomatic FAP, 30 who were asymptomatic, and 8 disease-free spouses. Twenty individuals with symptomatic FAP were all heterozygous for the prealbumin genes, carrying one normal and one mutant gene. We confirmed the direct linkage between the mutant prealbumin gene and the Japanese type of FAP. Moreover, 10 of the 30 asymptomatic individuals from pedigrees with FAP were also heterozygous for the prealbumin gene. The number of asymptomatic individuals with the mutant prealbumin gene showed age-related decreases, and none was over 40 years. A linkage between the mutant prealbumin gene and serum levels of the prealbumin variant was also evident.     

Characterization of the complement inhibitory function of rhesus rhadinovirus complement control protein (RCP)

Rhesus rhadinovirus (RRV) is currently the closest known, fully sequenced homolog of human Kaposi sarcoma-associated herpesvirus. Both these viruses encode complement inhibitors as follows: Kaposi sarcoma-associated herpesvirus-complement control protein (KCP) and RRV-complement control protein (RCP). Previously we characterized in detail the functional properties of KCP as a complement inhibitor. Here, we performed comparative analyses for two variants of RCP protein, encoded by RRV strains H26-95 and 17577. Both RCP variants and KCP inhibited human and rhesus complement when tested in hemolytic assays measuring all steps of activation via the classical and the alternative pathway. RCP variants from both RRV strains supported C3b and C4b degradation by factor I and decay acceleration of the classical C3 convertase, similar to KCP. Additionally, the 17577 RCP variant accelerated decay of the alternative C3 convertase, which was not seen for KCP. In contrast to KCP, RCP showed no affinity to heparin and is the first described complement inhibitor in which the binding site for C3b/C4b does not interact with heparin. Molecular modeling shows a structural disruption in the region of RCP that corresponds to the KCP-heparin-binding site. This makes RRV a superior model for future in vivo investigations of complement evasion, as RCP does not play a supportive role in viral attachment as KCP does.